Although many life-threatening drug reactions are reported each year, very little is known about their etiologies. We believe that many of these toxicities might be caused by the covalent alterations of specific macromolecules by reactive metabolites of drugs. These modifications might lead to toxicity directly by changing the physiological function of macromolecules or indirectly by making the macromolecules immunogenic and therefore causing immune-mediated toxicities. All of our studies have been directed toward studying these possible mechanisms of toxicity, with an ultimate goal of being able to design safer drugs. This year it was found that the processing of the trifluoroacetylated liver neoantigens associated with halothane hepatitis could be studied in primary cultures of rat liver cells. It was found that under normal conditions the parenchymal cells of the liver and not the Kupffer or endothelial cells were the major sites of turnover of the neoantigens. It appeared that a cellular serine protease was responsible, at least in part for the turnover of the neoantigens. Very little, if any of the neoantigens were excreted from the hepatocytes. In order to learn more about the molecular basis of the antigenicity and physiological function of the 58 kDa antigen associated with halothane hepatitis, we have started to clone it from from a human liver cDNA library and to determine its cellular location in various organs of the body by immunohistochemical techniques. Several cDNA clones have been isolated and are currently being sequenced. The immunohistochemical studies have revealed that the 58 kDa protein is concentrated in specific cells of several organs. For example, it is expressed predominantly in the ganglionic cells of the heart. We have continued to study the mechanisms of formation of protein adducts of nonsteroidal antiinflammatory agents, the most widely used class of drugs. Immunofluorescence studies of intact primary hepatocytes have confirmed that the major 110 kDa liver target of diclofenac resides in the plasma membrane adduct.